The stable transmission of genetic material is the foundation of life reproduction. The precise replication and distribution of DNA is the basis of genetic material transfer, and it is also the two core biological events of the cell cycle. DNA-related enzymes are one of the most important factors in the replication process. They are involved not only in the process of normal genomic DNA synthesis, but also in various repair processes under DNA damage. A variety of DNA-related enzyme gene mutations are associated with certain tumors and genetic diseases. DNA-related enzyme electrochemical biosensors make detection easier. 3-(N-Morpholino)propanesulfonic acid (MOPS) is a commonly used biological buffer.

Recently, researchers [1] have provided a DNA-related enzyme electrochemical biosensor based on DNA-copper nanoclusters with good specificity, high sensitivity, fast detection speed, accurate and reliable results, and low cost. The specific steps are as follows:

(1) Electrode 1: Mix sulfhydryl DNA (2.5~7.5 μL, 0.25~0.75 μM) and tris(2-carboxyethyl)phosphine (TCEP) (0.5~1.5 μL, 0.5~1.5 mM), evenly at 27~42°C for 30~90 min, drop on the surface of a clean bare gold electrode (Au), and wash the electrode with dilute water.
(2) Electrode 2: Take 1~3 μL of MOPS buffer (10 mM MOPS, 150 mM NaCl, pH 7.6), 0.5 to 1.5 μL of 5×TdT buffer, and dTTP (0.5~1.5 μL, Prepare TdT reaction solution with 5~15mM) and TdT (0.1~1.5μL, 5~15U/mL), control the total volume to 2.5~7.5μL, mix well, drop on the surface of Electrode 1 and place at 27~42°C 0.5~1.5h, wash the electrode with dilute water.

(3) Electrode 3: Cu2+ (2.5~7.5 μL, 0.5~1.5 mM) was added dropwise to the electrode surface of Electrode 2, and incubated at room temperature for 10~30 minutes, and the electrode was slowly buffered by distilled water. Then, sodium ascorbate solution (2.5~7.5 μL, 1~3 mM) was added dropwise to the surface of the electrode, and reacted at room temperature for 10~30 minutes, and the electrode was slowly washed by distilled water.

Compared with the prior process, the sensor has the following advantages:
(1) High sensitivity. The current response of the sensor to the TdT detection limit is 0.0008U/mL, the detection limit of Exo I concentration is 0.002U/mL, indicating that the sensor achieves highly sensitive detection of TdT and Exo I.
(2) High specificity. Other common enzymes such as urease (Urease), acetylcholinesterase (AChE), uracil-DNA glycosylase (UDG), histone acetylase (HAT), protein kinase (PKA), alkaline phosphatase (ALP), has no interference to the TdT and Exo I activity detection systems.
(3) Inhibitor detection. Through the electrochemical response of the electrochemical biosensor to the enzyme activity, the detection of the TdT inhibitor sodium pyrophosphate (PP) and the Exo I inhibitor polyethylene glycol (PEG) can be achieved, and the correlation of electrochemical response of the sensor with enzyme inhibitors can be obtained..
(4) The reagents are in a small amount, the detection speed is fast, and the cost is low.

In summary, the DNA-related enzyme electrochemical biosensor has the advantages of high sensitivity, good selectivity, simple operation, rapid analysis and easy operation, and can realize detection of low concentration TdT and Exo I, and has good application prospect.

Reference

[1] DNA-related enzyme electrochemical biosensor based on DNA-copper nanocluster and its application. CN109613096A, 2019-01-21.

Edited by Suzhou Yacoo Science Co., Ltd.

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